Fig. 3

Inhibition of oxidative stress ameliorates MMA-mediated cardiomyocytes injury. In vitro, AC16 cells were treated with MMA (17.5 mM) alone for 3 h or with Acetylcysteine (NAC) (5 mM) for 2 h followed by MMA (17.5 mM) for 3 h. In vivo, mice were assigned to the sham operation or myocardial I/R injury group. MMA (400 mg/kg/d) was administered seven days before the I/R injury. Meanwhile, NAC (300 mg/kg) was administered three times before the I/R model (on day 2, 4, and 6 of the administration process). a-c NAC (5 mM) inhibited the expression of NOX2 and NOX4 in AC16 cells observed by confocal microscopy (n = 3–4/group) (Scale bar: 50 μm). d-f Immunofluorescence staining analysis to assess the NOX2 and NOX4 expression (n = 3/group) (Scale bar: 100 μm). g, h Western blotting analysis to determine NOX4 and NOX2 protein expression in the heart (n = 3/group). i, j Western blot assessed the NOX4 and NOX2 protein level in AC16 cells (n = 3/group). k NAC inhibited fluorescence intensity of DHE shown by Flow cytometry (n = 3/group). l, m Representative photomicrographs and averaged data of TUNEL positive cells (n = 4/group) (Scale bar: 50 μm). n NAC relieved myocardial damage assessed by CK-MB assay (n = 4/group). Data are expressed as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and n.s, not significant